Urease induced calcium precipitation by Helicobacter species may initiate gallstone formation

Helicobacter species can colonise the mammalian gastrointestinal and hepatobiliary tract which usually results in a chronic infection coupled to an inflammatory host response. It is therefore not surprising that colonisation with Helicobacter species is linked with a range of inflammation associated gastrointestinal and hepatobiliary diseases.¹ Recently, this range has been expanded, with an association of infection with enterohepatic Helicobacter species and the formation of cholesterol gallstones.²

In their study, Maurer and colleagues² demonstrated that murine infection with the enterohepatic Helicobacter species H bilis and H hepaticus accelerated the formation of cholesterol gallstones in mice fed a lithogenic diet. Although the gallbladder mucosa in mice with gallstones displayed signs of inflammation, Helicobacter species were not cultured from the inflamed gallbladder or bile. Therefore, Maurer et al hypothesised that the chronic immune stimulation caused by Helicobacter species, rather than a direct bacterial factor, led to the production of nucleating agents, thus indirectly linking Helicobacter species and cholesterol gallstone formation.² Although a role for inflammation cannot be excluded, we believe that identification of Helicobacter DNA in gallstones³,⁴ is an indication that Helicobacter species may also play a direct role in gallstone formation.

Gallstones are crystalline bodies formed by accretion or concretion of bile components. Approximately 80% of gallstones are cholesterol gallstones, and 20% are pigment stones consisting of bilirubin and calcium, two components present in bile. There is a connection between the two types of gallstones as calcium‐bilirubin salts form surfaces that are highly attractive for cholesterols to adhere to.⁵ In addition, almost all types of gallstones contain a nidus of calcium.⁶,⁷ Bacteria have been shown to be able to initiate calcium precipitation and subsequent stone formation,⁸,⁹ similar to Proteus mirabilis and kidney stones.¹⁰ An important factor in bacteria induced stone formation is the enzyme urease which hydrolyses urea into ammonia and bicarbonate. Ammonia increases the pH and thereby favours generation of an insoluble form of calcium and subsequent precipitation, and this may be a mechanism shared between different types of stones.

We noticed in the study of Maurer and colleagues² that cholesterol gallstone formation was only detected after infection with the urease positive species H hepaticus and H bilis but not with the urease negative species H rodentium or H cinaedi.² This prompted us to investigate whether Helicobacter urease activity is involved in precipitation of calcium. For this purpose we developed a precipitation agar that allows for simultaneous growth of Helicobacter species and testing of their ability to precipitate calcium. We tested four different calcium concentrations (30, 10, 5, and 1 mM CaCl₂); best results were seen at 30 mM of calcium (fig 1) but calcium precipitation also occurred at more physiologically relevant calcium concentrations (10, 5, and 1 mM)¹¹ although the crystals were smaller. All four urease positive Helicobacter species tested (H hepaticus, H bilis, H pylori, and H mustelae) were capable of precipitating calcium in our assay (fig 1) whereas isogenic urease negative mutants of three species as well as urease negative Helicobacter species (H pullorum and H cinaedi) were unable to do so (fig 1). Purified urease enzyme alone was also capable of precipitating calcium.

Figure 1 Urease activity is required for calcium precipitation by Helicobacter species. Top panel: Comparison of H hepaticus strain ATCC51449, H pylori strain 26695, and H mustelae strain NCTC12198 (left) and their isogenic urease negative mutants (right). Bottom panel: Comparison of wild‐type H bilis, H pullorum, and H cinaedi isolates. All Helicobacter species were grown on precipitation agar (Columbia agar supplemented with Dent selective supplement (Oxoid), 0.2% β‐cyclodextrins, 333 mM urea, 100 μM NiCl₂, 20 mM NaHCO₃, 187 mM NH₄Cl, 30 mM CaCl₂, and 0.04% triphenyl tetrazolium chloride; all concentrations given are final concentrations). After overnight growth under microaerobic conditions (5% O₂, 10% CO₂, 85% N₂), calcium crystals were visible with urease positive Helicobacter species but not with urease negative Helicobacter species or with the isogenic urease negative mutants. Magnification 3×, photos taken with three megapixel digital camera. The figure represents a representative example of three independent experiments.

This suggests that urease positive Helicobacter species that are able to survive in or colonise the bile ducts (which excludes H pylori¹²) may induce the formation of gallstones both directly via their urease activity and indirectly via the immune response. Our observations extend those previously reported,²,³,⁴,¹² and provide a possible mechanism to explain the association between hepatobiliary colonisation with urease positive Helicobacter species and gallstone formation.